Isolation of Isoflavones from Iraqi Trifolium pretense

Noor Mohsen Naseer¹, Talal A. Aburjai², Ibrahim S. Al-Jubori¹

¹College of Pharmacy, Mustansiriya University, Baghdad, Iraq.

²School of Pharmacy, The University of Jordan.

*Corresponding Author E-mail: aburjai@ju.edu.jo, noor_mohsen2016@uomustansiriyah.edu.iq

ABSTRACT:

Objective: Trifolium pretense considered one of the most important medicinal plant which belongs to Fabaceae family, this plant commonly known as Red clover, its native region is Europe, Western Asia, and northwest Africa, but planted and naturalised in many other regions. The main compoundes found in Trifolium pretense are isoflavones which is determinrd by genistein, daidzein, biochanin A and formononetin. These compounds structurally related to estradiol-17 beta. Mangement of cancer is the main medicinal uses of Trifolium pretense in addition to decrease cholesterol and LDL and had anti- inflammatory activity, antioxidant and its effect on the Skin, Appendages, and Mucosal Status in Postmenopausal Women. This study was designed to isolate the main isoflavones from Trifolium pretense. Methods: The plant was collecting and washed by tap water to remove dirt and dust and rinsed with distilled water then dried at room temperature. Then, the plant was extracted by ethanol in a soxhlet apparatus and the extract partitioned with ethyl acetate by separatory funnel. The ethyl acetate layer was subjected to standard methods for active constituents identification, and purified by column chromatography. The collected fractions were analysed by TLC comparing with standard isoflavones (genistein, daidzein, biochanin A and formononetin). Preparative TLC was performed using 0.5 mm thickness of silica gel; the isolated bands were conducted with AR grade acetone. Iisolated isoflavones were characterized by ¹H and ^13-CNMR. Results: Isolated isoflavones (genistein, daidzein, biochanin A and formononetin) were identified by melting points which were identical with to that which were reported in the literature. Moreover, the ¹H-NMR and ¹³C-NMR analysis were used to identify the target compounds in CDCl₃ solvent, and the values of chemical shifts have been discussed according to the literature of analogous compounds.

KEYWORDS: Trifolium pretense, Red clover, Isoflavones, estradiol-17 beta, genistein, daidzein, biochanin A and formononetin.

INTRODUCTION:

History of using plants in the treatment of diseases is very ancient that belong to about sixty thousand years ago in every culture and all countries¹. Trifolium pretense considers one of the most important medicinal plant which belongs to Fabaceae family and, commonly known as Red clover. Its native regions are Europe, Western Asia, and northwest Africa, but planted and naturalised in many other regions². The plant has gained popularity, as a rich source of isoflavones such as genistein, daidzein, biochanin A and formononetin^3-5.

These compounds are phenols with heterocyclic ring and have similar structural to estradiol-17 beta as well as considered as selective estrogen receptor modulators^6,7. These isoflavones had many medicinal activities since and are considered as phytoestrogen since their structures are closely related to estrogen^8,9. Isoflavones and flavonoids are important phenolic secondary metabolite in plants with significant chelating and antioxidant properties^10-12. These compounds are based on the flavone nucleus, in which the number, positions, and types of substituent influence radical scavenging and chelating activity. Most From many medical activities are the effect on the skin such as, appendages, and mucosal status in postmenopausal women¹³, anticancer activity^14,15, decreasing of cholesterol and LDL levels¹⁶and had anti-inflammatory activity^17,18.

MATERIALS AND METHODS:

Plant materials:

Aerial part of Trifolium pretense were obtained from Baghdad, and classified by Iraqi National Herbium in Abu-Ghraib. The plant were washed by tap water to remove all dirt and dust, rinsed with distilled water and dried at room temperature.

Preparation of plants extract and isolation of the isoflavones (genistein, daidzein, biochanin A and formononetin):

25gm of the Red clover were placed in thimble and extracted by a soxhlet apparatus using 250mL of 95% ethanol for 6 hrs till exhaustion. Then, ethanolic extract (TE) was concentrated under vacuum using rotary evaporator and was tested by thin layer chromatography (TLC) comparing with standards in three different mobile phases. (TE) extract. After that, TE was partitioned three times with ethyl acetate (3 x 30mL) using separatory funnel and the ethyl acetate layers were combined, dried with anhydrous sodium sulphate, filtered and evaporated under vacuum¹⁹.

Column chromatography:

The ethyl acetate layer was purified by column chromatography using toluene: ethyl acetate: formic acid as mobile phase. The collected fractions were analysed by TLC comparing with isoflavones standards (genistein, daidzein, biochanin A and formononetin).

Thin layer Chromatography:

The retention factor (Rf value) of isolated isoflavones (genistein, daidzein, biochanin A and formononetin) were compared with Rf values of standards in three different solvent systems²⁰. Preparative TLC plates were performed using 0.5mm thickness of silica gel. The isolated bands were washed and extracted by AR grade acetone. Isolated isoflavones were identified by ¹H and ¹³C-NMR.

4′, 5, 7-Trihydroxyisoflavone (Genistein):

Genistein

¹H NMR (500 MHz, DMSO-d6) δ ppm: 6.23 (s, 1H, H-6), 6.39 (s, 1H, H-8), 6.82 (d, 2H, H-3′,5′), 7.38 (d, 2H, H-2′,6′), 8.33 (s, 1H, H-2), 9.61 (s, 1H, OH-4′), 10.91 (s, 1H, OH-7), 12.97 (s, 1H, OH-5). ¹³C NMR (MHz, DMSO-d6) δ ppm: 93.6 (C-8), 98.8 (C-6), 104.3 (C-10), 114.9 (C-3′, 5′), 121.1 (C-3), 122.2 (C-1′), 130.1 (C-2′, 6′), 153.9 (C-2), 157.3 (C-9), 157.5 (C-4′), 161.9 (C-5), 164.2 (C-7), 180.1 (C-4).

7-Hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (Daidzein):

Daidzein:

¹H NMR (500 MHz, DMSO-d6) δ ppm: 6.94 (s, 1H, H-6), 6.81 (d, 2H, H-3′, 5′),6.86 (s, 1H, H-8), 7.38 (d, 2H, H-2′, 6′), 8.29 (s, 1H, H-2), 9.52 (s, OH-4′)10.99 (s, OH-7),7.97 (s, OH-5).¹³C NMR (125 MHz, DMSO-d6) δ ppm: 102.0 (C-8), 114.9 (C-6), 116.6 (C-10), 118 (C3′, 5′), 122.1 (C-3), 123.2 (C-1′), 130.1 (C-2′, 6′), 152.8 (C-2),157.3 (C-9), 157.5 (C-4′), 127.3 (C-5), 162.2 (C-7), 174.7 (C4).

7-Hydroxy-3-(4-methoxyphenyl) chromen-4-one (Formononetin):

Formononetin

¹H-NMR(500 MHz, DMSO-d6) δ ppm: 3.79 (s, 3H, OCH3), 6.87 (d, 1H, H-8), 6.95 (dd, 1H, H-6), 6.97 (d, 2H, H-3', 5'), 7.50 (d, 2H, H-2', 6'), 7.98 (d, 1H, H-5), 8.35 (s, 1H, H-2), 10.81 (s, 1H, OH). ¹³C-NMR (125 MHz, DMSO-d6) δ ppm: 102.1(C-8), 113.6 (C-3', 5'), 115.2 (C-6), 116.6 (C-10), 123.1(C-1'), 124.2 (C-3), 127.3 (C-5), 130.1 (C-2', 6'), 153.2 (C-2), 157.4 (C-9), 158.9 (C-4'), 162.6 (C-7), 174.6 (C-4).

5,7-Dihydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one (Biochanin A):

Biochanin A

¹H-NMR(500 MHz, DMSO-d6) δ :12.93 (s, 1H, OH-5), 10.81 (s, 1H, OH-7), 8.38 (s, 1H, H-2), 7.51 (d, 2H, H-2',6'), 7.01 (d, 2H, H-3', 5'), 6.40 (d, 1H, H-8), 6.24 (d, 1H, H-6), 3.79 (s, 3H, OCH3). ¹³C-NMR(125 MHz, DMSO-d6) δ ppm: 93.7 (C-8), 99.0 (C-6), 104.5 (C-10), 113.7 (C-3', 5'), 122.0 (C-1'), 122.9 (C-3), 130.2 (C-2', 6'), 154.3 (C-2), 157.6 (C-9), 159.2 (C-4'), 162.0 (C-5), 164.3 (C-7), 180.1 (C-4).

RESULTS AND DISCUSSION:

The presence of isoflavones in the plant was confirmed by TLC of (TE) extract by comparing with standards in 3 three mobile phase, toluene: ethyl acetate: formic acid (3: 2: 0.3), toluene: chloroform: acetone (4: 2.5: 3.5), toluene: ethyl acetate: chloroform (5: 3: 4). Table 1 shown the Rf values of isoflavones in three mobile phases S1, S2, and S3. (Figure 1) displays TLC plates of studied isoflavones in TE.

Table1. Rf of standards and isolated isoflavones in three solvent systems.

Solvent system	R_f of genistein stanrad	R_f of isolated genistein	R_f of daidzein standard	Rf of isolatd daidzein	R_f of biochanin standard	R_f of isolated biochanin A	R_f of formononetin standard	R_fof isolated formononetin
(S1) toluene:ethyl acetate: formic acid (3: 1: 0.3)	0.5	0.49	0.25	0.25	0.73	0.72	0.56	0.56
(S2) toluene: chloroform: acetone (4: 2.5: 3.5)	0.57	0.57	0.47	0.48	0.75	0.74	0.68	0.68
(S3) toluene: ethyl acetate: chloroform (5: 3: 4)	0.53	0.52	0.24	0.25	0.75	0.75	0.6	0.6

S1 S2 S3

Figure1. TLC analysis of ethanolic extract

The presence of isolated isoflavones was confirmed by PTLC by comparing with standards as shown in (Figure 2) ²¹.

A): Daidzein (B): Genistein and& Formononetin (C): Biochanin A

Figure 2: PTLC of isolated isoflavones by comparing with standards. A: Daidzein; (B): Genistein & Formononetin; (C): Biochanin A.

Isolated isoflavones were identified by melting points which were (301, 315, 215 and 195°C) respectively, which wereas identical with that reported in the literature^22-24.

¹H-NMR analysis were used to identify the target compounds in CDCl3 solvent and spectra for all isolated compounds displayed common resonances in the ranges of 8.29-8.38, 6.23-6.95, 6.39-6.87, 7.38-7.51, and 6.81-7.01 ppm for protons H-2, 6, 8, 2', 6', 3', and 5', respectively. Table 2 summarizes the common peaks for all isolated isoflavones and the spectra of formononetin compound were displayed in Figures 3. The spectra of the other derivatives are available in Supplementary Data. The values of chemical shifts were comparable to the values of same compounds that previously mentioned in literatures.

Table 2: Important protons in 1H-NMR spectra of isolated isoflavones.

Seq.	Compounds	¹H-NMR Chemical shifts (δ, ppm)
Seq.	Compounds	H-2	H-6	H-8	H-2', 6'	H-3', 5'
1	Genistein	8.33	6.23	6.39	7.38	6.82
2	Daidzein	8.29	6.94	6.86	7.38	6.81
3	Formononetin	8.35	6.95	6.87	7.50	6.97
4	Biochanin A	8.38	6.24	6.40	7.51	7.01

(A): ¹H-NMR (B): ¹³C-NMR

Figure 3: 1H-NMR and& 13C-NMR spectra of Formononetin.

The ¹³C-NMR spectra for these isoflavones showed common resonances in the ranges of 152.8-154.3, 121.1-124.2, 99.0-115.2, 93.6-94.1, 130.1-130.9, and 113.6-114.9 ppm for carbons C-2, 3, 6, 8, 2', 6', 3', and 5', respectively, which confirm the structure identification of the studied compounds. Table 3 summarizes the common peaks for all isolated isoflavones and the spectra of formononetin compound were displayed in Figures 3. The spectra of the other derivatives are available in Supplementary Data.

Table 3: Important protons in ¹³C-NMR spectra of isolated isoflavones.

Seq.	Compounds	¹³C-NMR Chemical shifts (δ, ppm)
Seq.	Compounds	C-2	C-3	C-6	C-8	C-2',6'	C-3',5'
1	Genistein	153.9	121.1	114.9	93.6	130.31	114.9
2	Daidzein	152.8	122.1	98.8	93.5	130.9	114.8
3	Formononetin	153.2	124.2	99.0	94.1	130.1	113.6
4	Biochanin A	154.3	122.9	115.2	93.7	130.2	113.7

The studied isoflavones compounds were analyzed using Fourier Transform Infraredspectroscopy (FTIR) to identify the functional groups that present in each particular derivative. FTIR spectra for all isoflavones family exhibit absorption bands around 3122-3408, 3076-3303, 2683-2914, 1605-1650, 1567-1594, 1243-1278, and 718-740 cm^-1 which belong to =C-H sp² stretching OH, -C-H sp³ stretching, C=O stretching, C=C aromatic stretching C-O stretching C-H aromatic, respectively, which confirm the identification of the isolated isoflavones as shown in Table 4. Figure 4 displays the FTIR spectrum of formononetin compound. FTIR spectra of the other derivatives are available in Supplementary Data.

Table 4: FTIR frequencies of the major functional groups in isolated isoflavones

Seq.	Compounds	Important FTIR frequencies Frequency/wavelength (ʋmax in cm^-1)
Seq.	Compounds	=C-H sp² Stretching	OH	-C-H sp³ Stretching	C=O stretching	C=C aromatic stretching	C-O stretching	C-H aromatic
1	Genistein	3408	3097	2914	1650	1567	1255	740
2	Daidzein	3215	3113	2683	1630	1594	1278	725
3	Formononetin	3122	3076	2721	1605	1594	1245	718
4	Biochanin A	3384	3303	2837	1650	1583	1243	722

Figure 4: FTIR spectrum of formononetin compound.

CONCLUSION:

The valuable Iraqi medicinal plant Trifolium pretense, contains isoflavones a major active constituents. Isoflavones are responsible for this plant in treatment of many medical problems, especially those which related to menopausal women, since they considered as phytoestrogens according to their chemical structures. Four isoflavones were isolated, purified and characterized by melting points, FTIR, ¹H and ¹³C-NMR analysis.

ACKNOWLEDGMENT:

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad- Iraq for its support in the present work.

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Received on 17.10.2021 Modified on 20.12.2021

Research J. Pharm. and Tech 2022; 15(10):4692-4696.

DOI: 10.52711/0974-360X.2022.00787